Use of Native Peptides and Their Optimized Derivatives For Vaccination

ABSTRACT

The present invention pertains to the field of vaccination, and more particularly to the fields of antitumor and antiviral vaccination. The invention relates to the use of a native peptide in a medicinal composition, for selecting and/or boosting part of a CTL immune response which has been initiated by an optimized immunogenic peptide derived from said native peptide. The invention also concerns vaccination kits which comprise several doses of optimized peptides and of their cognate native peptides.

This application is a continuation of U.S. application Ser. No.11/913,138, filed Nov. 18, 2007, which is a national stage applicationfiled under 35 U.S.C. 371 of International Application NoPCT/EP2006/005325, filed May 9, 2006, which claims priority fromEuropean patent Application No. 05290984.3, filed May 9, 2005, thecontents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention pertains to the field of vaccination, and moreparticularly to the fields of antitumor and antiviral vaccination. Theinvention concerns the use of a native peptide in a medicinalcomposition, for selecting and/or boosting part of a CTL immune responsewhich has been initiated by an optimized immunogenic peptide derivedfrom said native peptide.

BACKGROUND OF THE INVENTION

Cancer immunotherapy is intended to stimulate cytotoxic T lymphocytes(CTL) recognizing peptides derived from tumor antigens and presented atthe tumor cell surface by HLA class 1 molecules. CTL targeted peptidescan be dominant or cryptic (Moudgil and Sercarz 1994). Dominant peptideshave high HLA affinity and are frequently presented by tumor cells. Incontrast, cryptic peptides have low HLA affinity and are rarelypresented by tumor cells. All cancer vaccines so far tested havetargeted dominant peptides, with relatively little success (Slingluff,Yamshchikov et al. 2001; Knutson, Schiffman et al. 2002; Schaed, Klimeket al. 2002; Parkhurst, Riley et al. 2004; Vonderheide, Domchek et al.2004). Studies using mouse models showed that this lack of efficacy isdue to tolerance to tumor antigens, and especially to their dominantpeptides (Cibotti, Kanellopoulos et al. 1992; Theobald, Biggs et al.1997; Colella, Bullock et al. 2000; Hernandez, Lee et al. 2000;Grossmann, Davila et al. 2001; Gross, Graff-Dubois et al. 2004).

To circumvent this tolerance, vaccination with cryptic peptides wasrecently proposed. It was observed that in humanized mice, tolerance ofcryptic peptides was weak or absent, and that cryptic peptidesefficiently induced antitumoral immunity in vivo, providing theirimmunogenicity had been optimized (Tourdot, Scardino et al. 2000;Scardino, Gross et al. 2002; Gross, Graff-Dubois et al. 2004). A peptidesequence modification that optimizes immunogenicity of almost alllow-affinity HLA-A*0201-restricted peptides tested was previouslydescribed (Tourdot, Scardino et al. 2000).

TERT_(572Y) is an HLA-A*0201-associated optimized cryptic peptidederived from TERT, an antigen overexpressed by 85% of human tumors (Kim,Piatyszek et al. 1994). TERT₅₇₂ is present in both human and murineTERT, and TERT_(572Y) was able to induce antitumoral immunity inHLA-A*0201 transgenic mice; however no autoimmunity against normalTERT-expressing tissues was observed (Gross, Graff-Dubois et al. 2004).In vitro, TERT_(572Y) stimulated antitumor CTLs from both healthy donorsand prostate cancer patients. CTLs killed TERT-expressing tumor cellsbut not TERT-expressing normal cells (Hernandez, Garcia-Pons et al.2002; Scardino, Gross et al. 2002).

However, in a similar vaccination approach, it has been reported thatvaccination of melanoma patients with optimized gp100_(209M) led to theamplification of T cells that were no longer able to recognize eitherthe native gp100₂₀₉ peptide or gp100-expressing melanoma cells (Clay,Custer et al. 1999).

Hence, there is presently a need for a vaccination protocol whichenables the initiation and maintenance of a T cell response targetingsub-dominant or cryptic epitopes, especially when this response isinitiated by optimized peptides.

SUMMARY OF THE INVENTION

The study disclosed in Example 1 below was designed to evaluate: i) thecapacity of TERT_(572Y) to stimulate an antitumor immune response invivo in patients with advanced cancer; and ii) the risk of inducingautoimmunity against TERT-expressing normal cells and tissues such ashematopoietic precursors, gut, thymus and liver. Vaccination of advancedcancer patients with TERT_(572Y) stimulated specific CTLs that werefully functional and able to kill in vitro tumor cells overexpressingTERT. Moreover, vaccination was safe and did not induce any autoimmunityagainst TERT positive normal tissues. This is the first in vivodemonstration in humans that optimized cryptic peptides can beconsidered for tumor immunotherapy.

Moreover, these results, as well as those presented in Examples 2, 3 and4, show that injection of a native peptide, following vaccination withits cognate optimized peptide, can maintain the immune responseinitiated by said optimized peptide. Without being bound by theory, itcan be hypothesized that the use of the native peptide allows to selectand/or boost, among T cells recruited by the optimized peptide, thosewith the highest specificity for the native peptide presented by tumorcells.

These findings allow to propose the use of a native cryptic ornon-optimized peptide for improving the CTL immune response raised by acognate optimized peptide.

A “cryptic peptide” for a given MHC molecule is a peptide which iscapable to bind said MHC molecule, but only with a weak affinity for theMHC molecule and/or a weak stability of the MHC/peptide complex. As aresult this peptide is only poorly presented by said MHC molecule at thesurface of an antigen presenting cell, and thus participate onlyslightly, or not at all, in the CTL response to the antigen from whichsaid peptide is derived. For example, in the case of HLA A2, crypticpeptides can be defined as peptides which have a low affinity and a weakstabilizing ability (RA>5 and DC₅₀<2 hours), as described in WO 0208716.

A “native peptide” (cryptic or not) is a peptide which corresponds to afragment of an antigen, without any sequence modification.

An “optimized peptide” for a given native peptide is a peptide obtainedby one or several amino acid substitutions in said native peptide, saidmodifications resulting in a greater affinity for the MHC moleculeand/or a greater stability of the MHC/peptide complex. For example,HLA-A2.1-associated peptides can be optimized by modifying theirsequence by introducing a tyrosine in the first position (P1Ysubstitution) (Tourdot, Scardino et al. 2000). A method for identifyingcryptic peptides and generating cognate optimized peptides is disclosedfor instance in PCT WO 02/08716, the content of which is incorporatedherein by reference. Other modifications for optimizing HLA A2 peptideshave also been described, such as substituting the amino acid inposition 2 by a methionine or a leucine (Parkhurst, Salgaller et al.1996; Bakker, van der Burg et al. 1997; Valmori, Fonteneau et al. 1998),or substituting the C-terminal amino acid by a valine or a leucine(Parkhurst, Salgaller et al. 1996). These peptide modifications can bedone to obtain optimized peptides to perform the present invention.

A cryptic peptide is not able to generate in vitro a specific CTLresponse against target cells expressing the protein from which it isderived. In contrast, the cognate optimized peptide is able to generatea specific CTL response against the same target cells, wherein at leastpart of the CTLs have a high avidity for said cryptic peptide.

An object of the present invention is the use of a native peptide, forproducing a medicinal composition for maintaining the CTL immuneresponse initiated by its cognate optimized peptide. According apreferred embodiment of the invention, the native peptide issub-dominant or cryptic.

The present invention is particularly useful in the domain of antitumoror antiviral immunotherapy. Accordingly, the native peptide isadvantageously from a tumor antigen or from a viral antigen, especiallyfrom an antigen from a virus which produces long-lasting infections,such as HIV, HCV and HBV.

According to the invention, a native peptide can be used for vaccinationof patients having previously received a cognate optimized peptide.

The present invention thus encompasses a method for vaccinating apatient against a tumoral or viral antigen, wherein said methodcomprises a first step of vaccination with an optimized peptide cognateto a native peptide of said antigen, particularly a cryptic peptide,followed by a second step of vaccination with said native peptide.

An example of antitumoral vaccination using the native cryptic peptideTERT₅₇₂ (RLFFYRKSV) and its cognate optimized peptide TERT_(572Y)(YLFFYRKSV), is given hereinafter in Example 1.

According to a preferred embodiment of the invention, the crypticpeptide is presented by HLA A2, and the optimized peptide results fromthe substitution of the N-terminal amino acid of said cryptic peptidewith a tyrosine residue. Non-limitative examples of couples of crypticpeptides and optimized peptides, presented by HLA A2, and which can beused in the present invention are described in PCT WO 02/08716 and inTable 1 below. Other couples of native and cognate optimized peptideswhich can be used according to the present invention, are also presentedin Table 1.

Another aspect of the present invention is a process for in vitroobtaining CTLs having high avidity for a native peptide, especially anative cryptic peptide, by stimulating, with said native peptide, theCTLs which are present in a biological sample from a patient who hasbeen immunized with a cognate optimized peptide. In this process, thenative and optimized peptides are advantageously as described above.

The present invention also pertains to a kit of parts for thevaccination, comprising at least one dose of a native peptide and atleast one dose of its cognate optimized peptide. In a preferredembodiment, the vaccination kit comprises 2 or 3 doses of optimizedpeptide, and 3, 4, 5 or 6 doses of native peptide. A particularvaccination kit according to the invention is adapted for the firstvaccination sequence of 6 injections, and comprises 2 or 3 doses ofoptimized peptide, and 4 or 3 doses of native peptide. In case oflong-lasting diseases, it is preferable to maintain the level ofimmunity obtained after this primo-vaccination, by regular recalls. Thiscan be done, for example, by injections performed every 3 to 6 months.Therefore, complementary kits, comprising at least 2 doses, and up to 40or 50 doses of native peptide, are also part of the present invention.Alternatively, the vaccination kit can comprise 2 to 3 doses ofoptimized peptide, and 3 to 40 or up to 50 doses of native peptide. Ofcourse, said native and optimized peptides present in the kit are asdescribed above.

Each dose comprises between 0.5 and 10 mg of peptide, preferably from 1to 5 mg. In a preferred embodiment, each dose is formulated forsubcutaneous injection. For example, each dose can be formulated in 0.3to 1.5 ml of an emulsion of aqueous solution emulsified with Montanide,used as an adjuvant. The skilled artisan can choose any otheradjuvant(s) in place of (or in addition to) Montanide. In a particularembodiment, the doses are in the form of an aqueous solution.Alternatively, the doses can be in the form of a lyophilized peptide,for extemporaneous preparation of the liquid solution to be injected.

TABLE 1examples of couples of native and optimized peptides, presented by HLA A2,which can be used according to the invention. Native peptideOptimized peptide Reference Name Sequence No Name Sequence No HIVgag₇₆SLYNTVATL 13 HIVgag_(76Y1) YLYNTVATL 14 WO 0208716 FluM₅₈ GIGLFVFTL 11FluM_(58Y1) YIGLFVFTL 12 HBVpol₅₇₅ FLLSLGIHL 15 HBVpol_(575y1) YLLSLGIHL16 HBVpol₇₆₅ LLGCAANWIL 17 HBVpol_(765Y1) YLGCAANWIL 18 Mart-1₂₇AAGIGILTV 19 Mart-1_(27Y1) YAGIGILTV 20 Mart-1₂₆ EAAGIGILTV 21Mart-1_(26L27) ELAGIGILTV 22 Valmori, D., 1998 Gp 100₁₇₇ AMLGTHTMEV 23Gp 100_(177Y1) YMLGTHTMEV 24 WO 0208716 Gp 100₁₇₈ MLGTHTMEV 25Gp 100_(178Y1) YLGTHTMEV 26 Gp 100₁₅₄ KTWGQYWQV 8 Gp 100_(154Y1)YTWGQYWQV 9 Gp 100_(154M155) KMWGQYWQV 10 Bakker, A.B., 1997 Gp 100₅₇₀SLADTNSLAV 27 Gp 100_(570Y1) YLADTNSLAV 28 WO 0208716 Gp 100₂₀₉ TDQVPFSV29 Gp 100_(209Y1) YDQVPFSV 30 Gp 100_(209M210) YMQVPFSV 31Parkhust, M.R., 1996 Gp 100₄₇₆ VLYRYGSFSV 32 Gp 100_(476Y1) YLYRYGSFSV33 WO 0208716 Gp 100₄₅₇ LLDGTATLRL 34 Gp 100_(457Y1) YLDGTATLRL 35HER-2/neu₇₉₉ QLMPYGCLL 36 HER-2/neu_(799Y1) YLMPYGCLL 37 HER-2/neu₃₆₉KIFGSLAFL 38 HER-2/neu_(369Y1) YIFGSLAFL 39 HER-2/neu₇₈₉ CLTSTVQLV 40HER-2/neu_(789Y1) YLTSTVQLV 41 HER-2/neu₄₈ HLYQGCQW 42 HER-2/neu_(48Y1)YLYQGCQW 43 HER-2/neu₇₇₃ VMAGVGSPYV 44 HER-2/neu_(773Y1) YMAGVGSPYV 45HER-2/neu₅ ALCRWGLL 46 HER-2/neu_(5Y1) YLCRWGLL 47 HER-2/neu₈₅₁VLVKSPNHV 48 HER-2/neu_(851Y1) YLVKSPNHV 49 HER-2/neu₆₆₁ ILLVVVLGV 50HER-2/neu_(661Y1) YLLVVVLGV 51 HER-2/neu₆₅₀ PLTSIISAV 52HER-2/neu_(650Y1) YLTSIISAV 53 HER-2/neu₄₆₆ ALIHHNTHL 54HER-2/neu_(466Y1) YLIHHNTHL 55 HER-2/neu₄₀₂ TLEEITGYL 56HER-2/neu_(402Y1) YLEEITGYL 57 HER-2/neu₃₉₁ PLQPEQLQV 58HER-2/neu_(391Y1) YLQPEQLQV 59 HER-2/neu₉₇₁ ELVSEFSRM 60HER-2/neu_(971Y1) YLVSEFSRM 61 HBVpol₂₈ LLDDEAGPL 62 HBVpo1_(28Y1)YLDDEAGPL 63 HBVpol₅₉₄ PLEEELPRL 64 HBVpol_(594Y1) YLEEELPRL 65HBVpol₉₈₅ NLQSLTNLL 66 HBVpol_(985Y1) YLQSLTNLL 67 EphA2₆₁ DMPIYMYSV 68EphA2_(61Y1) YMPIYMYSV 69 WO 03091383 HER2₉₁₁ TVWELMTFGA 70HER_(911Y1V10) YVWELMTFGV 71 HER4₉₁₁ TIWELMTFGG 72 HER1₉₁₁ TVWELMTFGS 73HER2₇₂₂ KVKVLGSGA 74 HER_(722Y1V9) YVKVLGSGV 75 HER3₇₂₂ KLKVLGSGV 76HER4₇₂₂ RVKVLGSGA 77 HER1₇₂₂ KIKVLGSGA 78 HER2₈₄₅ DLAARNVLV 79HER_(845Y1) YLAARNVLV 80 HER3₈₄₅ NLAARNVLL 81 HER2₉₀₄ DVWSYGVTV 82HER_(904Y1) YVWSYGVTV 83 WO 03083124 HER4₉₀₄ DVWSYGVTI 84 HER2₉₃₃DLLEKGERL 85 HER_(933Y1) YLLEKGERL 86 HER1₉₃₃ SILELKGERL 87 HER2₉₄₅PICTIDVYMI 88 HER_(945Y1) YICTIDVYMV 90 HER3₉₄₅ QICTIDVYMV 89 HER4₉₄₅PICTIDVYMV 91 HER1₉₄₅ PICTIDVYKI 92 MAGE-A_(248G9) YLEYRQVPG 7MAGE-A_(248V9) YLEYRQVPV 6 MAGE-A_(248D9) YLEYRQVPD 5 TERT₉₈₈ DLQVNSLQTV3 TERT_(988Y1) YLQVNSLQTV 4 WO 0208716 TERT₅₇₂ RLFFYRKSV 1 TERT_(572Y1)YLFFYRKSV 2

The invention is further illustrated by the following figures andexamples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: TERT_(572Y)-specific CD8 cells detected ex vivo in patients #1,#3, #8, #11 and #13. Thawed PBMC from patients #1, #8, #11 and #13collected before vaccination and after the second (#1, #8, #11) andsixth (#13) vaccine injections were stained with PE-labeled TERT_(572Y)tetramer, APC-labeled anti-CD8 and FITC-labeled anti-CD3. CD3+ gatedcells were analyzed.

FIG. 2: TERT_(572Y)-specific CD8 cells detected after in vitrostimulation of PBMC from patients #6 and #18. Thawed PBMC from patients#6 and #18 were cultured in the absence (unstimulated) or presence(stimulated) of 10 μM TERT_(572Y) for nine days. Cells were then stainedand analyzed as described in the legend of FIG. 1.

FIG. 3: Time course of immune responses.

FIG. 4: Functional analysis of TERT_(572Y)-specific CD8 cells induced byvaccination:

-   -   A) PBMC from patient #4, collected three weeks after the second        vaccine injection, were stimulated in vitro with TERT_(572Y)        peptide for nine days. TERT_(572Y) specific cells were purified        and amplified with PHA. Amplified cells were stained with        TERT_(572Y) tetramer and CD8 mAb.    -   B) TERT_(572Y) tetramer-positive cells were stimulated with        TERT_(572Y) and irrelevant FluM58 peptides for 6 hours, then        stained with PE-labeled anti-CD107a, permeabilized with Saponin        and stained with FITC-labeled anti-IFNγ to evaluate        intracellular IFNγ.    -   C) TERT_(572Y) tetramer-positive cells were incubated with        ⁵¹Cr-labeled N418 and TERT-transfected N418 cells for four hours        in a classical ⁵¹Cr release assay. E/T ratios are indicated.    -   D) TERT_(572Y) tetramer-positive cells were incubated with        ⁵¹Cr-labeled NA8 and ME290 tumor cells for four hours in a        classical ⁵¹Cr release assay. E/T ratios are indicated.

EXAMPLES Example 1 Safety and Immunogenicity of the Optimized CrypticPeptide TERT_(572Y) in Patients with Advanced Malignancies: Phase IClinical Study 1.1. Patients and Methods Patients

Patients with chemotherapy-resistant malignant tumors were eligible forthe study. Other eligibility criteria were: progressive disease forwhich there was no other therapeutic option of proven benefit; anexpected survival of at least 6 months; patients had to be HLA-A*0201positive; age 18-75 years old, performance status (WHO)<2, adequate bonemarrow (absolute neutrophil count≧1500/mm³; absolute lymphocytecount≧1300/mm³; platelets>100000/mm³; Hgb>10 g/dl), renal(creatinine<1.5 mg/dl) and liver (bilirubin<1.5 times the upper normalvalue) function. Patients were excluded if they had receivedchemotherapy, radiotherapy, hormonotherapy, immunotherapy orcorticosteroids within one month before enrolment or if they had a knownimmunodeficiency or auto-immune disease. The protocol had been approvedby the Ethics and Scientific Committees of the University Hospital ofHeraklion and the National Drug Administration of Greece. All patientsgave written informed consent in order to participate in the study.

Peptide Vaccine Preparation

The vaccine consisted of optimized TERT_(572Y) (YLFFYRKSV) and nativeTERT₅₇₂ (RLFFYRKSV) peptides emulsified in Montanide ISA51 (Seppic Inc,France). The vaccine peptides were synthesized at the Faculty ofPharmacy, University of Patras (Greece) by means of solid-phase Fmoc/Buchemistry. Quality assurance studies included confirmation of identity,sterility and purity (>95% for both peptides). No decrease in purity orconcentration was observed after more than two years of storage at −80°C. Each peptide was prepared as a lyophilized powder for reconstitutionand dilution in sterile water.

Vaccination Protocol

Patients received a total of six subcutaneous vaccinations administeredevery 3 weeks. Peptides in 0.5 ml aqueous solution were emulsified with0.5 ml Montanide ISA51 immediately before being injected. The optimizedTERT_(572Y) peptide was used for the first 2 vaccinations and the nativeTERT₅₇₂ peptide for the remaining 4 vaccinations. Five dose levels ofthe peptides were studied; dose levels included 2, 3, 4, 5 and 6 mg ofboth peptides. Three patients were entered at each dose level. Anadditional 3 patients were planned to be enrolled at the dose levelwhere a dose-limiting event was observed. Each patient received the samepeptide dose for all six vaccinations. No other treatment with possibleantitumor activity, i.e., chemotherapy, radiotherapy, hormonal therapyor administration of corticosteroids, was allowed during the course ofvaccination.

Patient Evaluation

Before entering the study, all patients were assessed by completemedical history, physical examination, and complete blood cell countwith differential, serum chemistry and baseline measurements of relevanttumor markers. Moreover, measurable disease was determined by standardimaging procedures (chest x-ray, ultrasound, computed tomography scansof thorax and abdomen, magnetic resonance imaging (MRI) if indicated,and whole body bone scans). Toxicity during the vaccination protocol wasevaluated by repeating the complete blood cell count weekly and byperforming medical history, physical examination and serum chemistryevery three weeks before each subsequent injection during thevaccination period and every month thereafter during the follow up.Toxicity was assessed and scored using the National Cancer Institute(NCI) Common Toxicity Criteria (Ajani, Welch et al. 1990). Dose-limitingtoxicity (DLT) was assessed during the entire vaccination protocol andwas defined as the occurrence of any of the following: grade 4hematologic toxicity; grade 3-4 neutropenia with fever>38.2° C.; grade3-4 non-hematologic toxicity; and any treatment delay because oftoxicity. Dose escalation was discontinued and the DLT dose level wasreached if at least 50% of the patients treated at that level develop aDLT. The MTD dose level was defined as the first level below the DLTdose level.

Response to treatment was evaluated by repeating the baseline imagingstudies and relevant tumor marker measurements after every 2vaccinations or sooner if clinically indicated. Response to treatmentwas scored as complete response (CR), partial response (PR), stabledisease (SD) and progressive disease (PD) using the standard WHOcriteria (Miller, Hoogstraten et al. 1981). Radiological responses wereconfirmed by an independent panel of radiologists. CR and PR had to bemaintained for a minimum of 4 weeks. The duration of response wasmeasured from the first documentation of response to diseaseprogression. Time to progression (TTP) was determined by the intervalbetween the initiation of therapy to the first date that diseaseprogression was objectively documented. Overall survival (OS) wasmeasured from the date of study entry to the date of death. The followup time was measured from the day of first treatment administration tolast contact or death. Immune responses were examined before the firstinjection, and after the second, fourth and sixth injections. Peripheralblood mononuclear cells (PBMC) were collected at each time point andfrozen.

Cell Lines

T2 is a mutant human TB hybrid that lacks TAP molecules but expressesHLA-A*0201. HLA-A*0201-positive N418 fibroblasts, TERT-transfected N418cells and the melanoma cell lines Nab and Me290 were provided by P.Romero (Ludwig Institute for Cancer Research, Lausanne, Switzerland).

Peptides

Class I-restricted peptides used for laboratory studies included TERT₅₇₂(RLFFYRKSV, SEQ ID No: 1), TERT_(572Y) (YLFFYRKSV, SEQ ID No: 2), andFluM₅₈ (GILGFVFTL, SEQ ID No: 11), all produced by Epytop (Nimes,France).

In Vitro Stimulation of PBMC

Thawed PBMC (3×10⁵ cells/well in 200 μl) were incubated in the presenceof 10 μM TERT_(572Y) peptide in complete medium (RPMI 1640 supplementedwith 8% human AB serum) in 96-well round-bottom plates. IL2 was added ata final concentration of 10 U/ml after 48 h and 96 h. Cells wereincubated at 37° C. in 5% CO₂-air. On day 9 of culture, cells from sixwells were pooled and analyzed for the presence of TERT_(572Y)-specificCD8 cells by TERT_(572Y) tetramer staining.

TERT_(572Y) Tetramer Staining

Cells were incubated with PE-conjugated TERT_(572Y) tetramer (ProimmuneLtd, Oxford, UK) for 30 min at room temperature, and then withAPC-conjugated anti-CD8 (BD Pharmingen, Mississauga, Canada) andFITC-conjugated anti-CD3 (BD Pharmingen, Mississauga, Canada) mAbs for30 min at 4° C. Stained cells were analyzed by flow cytometry(FACSCalibur, BD Biosciences, Mountain View, Calif.).

Polyclonal Expansion of TERT_(572Y) Tetramer-Positive Cells

PBMCs were stimulated with 10 μM TERT_(572Y) in the presence of 10 U/mlIL2 for 9 days. Cells were labeled with anti-CD8 mAb and TERT_(572Y)tetramer before isolation with a cell sorter. Sorted cells werestimulated with PHA (Difco) for 14 days.

CD107 and Intracellular IFNγ Double Labeling

T cells were stimulated with T2 cells loaded with peptide (10 μM) in thepresence of 20 μg/ml Brefeldin A (Sigma, Oakville, Canada). Six hourslater they were washed, stained with PE-conjugated anti-CD107 mAb (BDPharmingen, Mississauga, Canada) in PBS for 25 minutes at 4° C., washedagain and fixed with 4% paraformaldehyde. The cells were permeabilizedwith PBS/0.2% Saponin/0.5% BSA (Sigma) and stained with APC-conjugatedanti-IFNγ mAb (BD Pharmingen, Mississauga, Canada) before flowcytometric analysis (FACSCalibur, BD Biosciences, Mountain View,Calif.).

Cytotoxicity Assay

Target cells were labeled with 100 μCi of ⁵¹Cr for 90 min, washed twice,and plated in 96-well round-bottom plates (3×10³ cells/well in 100 μl ofRPMI 1640 plus 5% fetal calf serum). Effectors cells (100 μl) were thenadded to each well. After 4 h, 100 μl of supernatant was collected andradioactivity was measured with a gamma counter. The percentage ofspecific lysis was determined as follows: lysis=(experimentalrelease-spontaneous release)/(maximum release-spontaneous release)×100.

1.2. Results Patient Characteristics, Vaccination, and ClinicalResponses

The characteristics of the 19 patients enrolled in the trial are shownin Table 2.

TABLE 2 Patient characteristics. NSCLC = non small cell lung cancer, S =surgery, CT = chemotherapy, HT = hormone therapy, RT = radiotherapy, IT= immunotherapy (IL2, INFα, PS = performance status, PD = progressivedisease, SD = stable disease Dose Previous of No of Clinical Survival PtAge Sex Cancer Stage PS treatment vaccine injections response (months)#1 73 M colorectal IV 1 7 lines 2 mg 4 PD  6.1 CT #2 75 F breast IV 1 5lines 2 mg 6 PD 27.6 CT #3 64 M melanoma IV 1 1 line CT 2 mg 6 PD  8.8+#4 60 M NSCLC IV 1 2 lines 3 mg 6 PD 22.8+ CT #5 71 M NSCLC IV 1 6 lines3 mg 4 PD  5.7 CT #6 73 F cervix IV 1 2 lines 3 mg 6 PD  5.3 CT #7 53 Mhead and IV 1 4 lines 4 mg 6 PD 15.1 neck CT #8 57 F colorectal IV 1 5lines 4 mg 6 PD 12.3 CT #9 66 M renal IV 1 1 line IT 4 mg 6 SD 11.1+ #1073 F colorectal IV 1 2 lines 5 mg 6 PD  4.6 CT #11 49 M NSCLC IIIb 0 3lines 5 mg 6 SD 17.5+ CT #12 45 F breast IV 1 2 lines 5 mg 6 SD 11.2+ CT#13 51 M renal IV 1 1 line CT, 6 mg 6 SD  6.9+ 1 line IT #14 61 Munknown IV 1 2 lines 6 mg 4 PD  8 origin CT #15 70 F colorectal IV 0 2lines 6 mg 6 PD 10.7+ CT #16 69 M prostate IV 1 2 lines 6 mg 6 PD 17+ CT1 line HT #17 69 F ovarian IV 1 8 lines 6 mg 6 PD 13.7+ CT #18 51 Fovarian IV 1 4 lines 6 mg 5 PD  6.4 CT #19 48 M esophagus IV 1 1 line CT6 mg 4 PD  4.4+

All but one patient (patient #11) had stage IV cancer with multiplemetastases mainly in the bones, liver and lung. They all had active andprogressive disease and had received several treatments, mainlychemotherapy, before entering the vaccination protocol. Three patientswere enrolled at dose levels 2, 3, 4 and 5 mg of the peptides, whileseven patients received the 6 mg dose. Five patients were withdrawn fromthe protocol after the fourth (#1, #5, #14 and #19) or fifth (#18)vaccine injection because of rapid disease progression. All fivepatients subsequently died within six months of disease progression. Theremaining 14 patients completed the vaccination protocol. The diseasestabilized in four (29%) patients (#9, #11, #12 and #13) and continuedto progress in 10 patients. The latter 10 patients subsequently receivedchemotherapy, and six of them are still alive. One (patient #11) of the4 patients whose disease stabilized for 9 months subsequentlyprogressed, while the other three patients still have stable disease(after 12 months for patients #9 and #12, and 9 months for patient #13)with no additional therapy after the end of vaccination.

Overall, after a median follow up of 10.7 months (range 4.4-27.6), ninepatients have died, all due to disease progression. The median time totumor progression was 4.2 months (range 2.3-11.2) and the median overallsurvival 15.2 months (range 4.4-27.6).

Toxicity and Adverse Events

No DLT was observed throughout the entire study and therefore the MTDdose level has not been reached (Table 3). Thirteen patients developedgrade I toxicity. It consisted of local skin reaction (11 patients),anemia (6 patients), thrombocytopenia (2 patients), fatigue (1 patient)and anorexia (1 patient). Except of local skin reaction, othertoxicities were most likely related to the disease rather than thevaccination. Grade I toxicities appeared early in the vaccinationcourse. Three patients developed grade II toxicity consisting of fatigue(3 patients), nausea (2 patients) and anorexia (2 patients). In patient#5 (NSCLC) fatigue and nausea appeared after the third vaccination anddisappeared two weeks later without any specific treatment. In patient#10 (colorectal cancer) fatigue, nausea and anorexia observed after thethird vaccination were felt to be due to the disease rather than thevaccination. This patient developed a fatal intestinal obstruction.Patient #18 had an extremely rapid progression of her disease andconsequently was taken off the protocol after the fourth vaccination.She died two months later. Specifically, no significant hematologic,renal, gastrointestinal or hepatic toxicity was observed although TERTis expressed in these normal cells and tissues. Patients were monitoredfor toxicity for a median of 10.7 months (range 4.4-27.6). Even aftercompleting or discontinuing the vaccination program patients werefollowed monthly for the occurrence of any delayed toxicity. However, nosigns or findings of delayed toxicity were observed.

TABLE 3 Toxicity Toxicity Patient Grade I Grade II Grade III/IV #1 no nono #2 Local skin no no #3 Anemia, local skin no no #4 Local skin no no#5 no fatigue, nausea no #6 Anemia, local skin, fatigue, no no anorexia#7 no no no #8 Thrombo/penia, local skin no no #9 Local skin no no #10no anorexia, fatigue, no nausea #11 thrombocytopenia no no #12 Anemia,local skin no no #13 Local skin no no #14 Local skin no no #15 no no no#16 Anemia no no #17 Anemia, local skin no no #18 Local skin, anemiaanorexia, fatigue no #19 no no no

Immune Responses

Peptide-specific CD8⁺ cells were detected in peripheral blood by triplestaining of PBMCs with TERT_(572Y) tetramer, anti-CD8 and anti-CD3 mAbs,both ex vivo and after 9 days of stimulation in vitro with TERT_(572Y)peptide. In a preliminary study, TERT_(572Y) tetramer labeled less than0.11% of CD8 cells in seven HLA-A*0201 healthy donors (mean 0.035±0.035,range 0.0-0.11%) (data not shown). The positivity cutoff for specificimmunity was therefore set at 0.14% (mean+3SD) Immune responses werestudied in 14 vaccinated patients (Table 4). Only one patient (#2)failed to respond to the vaccine. TERT_(572Y)-specific cells weredetected ex vivo in four (29%) patients (FIG. 1). Specific immunityappeared after the second injection in patients #1, #8 and #11, andafter the sixth vaccination in patient #13. It is noteworthy that, priorto vaccination, TERT_(572Y) tetramer labeled 0.29%, 0.33% and 1.00% ofCD8⁺ cells in patients #1, #8 and #11 after in vitro PBMC stimulation(Table 4). TERT_(572Y)-specific cells were also detected in 9 patients(64%) after in vitro stimulation, 3 weeks after the 2^(nd) (#3, #4, #5,#6, #12, #15, and #19) or the 4^(th) (#7 and #18) injection.Representative results (patients #6 and #18) are shown in FIG. 2. Immuneresponse was also measured 3 and 14 months after the end of thevaccination protocol in patients #13 and #11 respectively. In all twopatients more than 1.5% of tetramer positive CD8 cells were detectedafter in vitro stimulation of their PBMC (FIG. 3).

TABLE 4 Percentage of tetramer TERT_(572Y)-positive CD8 cells amongperipheral blood mononuclear cells of vaccinated patients. % abovebackground in bold After the 2^(nd) or 4^(th) Pre-vaccination injectionPost-vaccination Patient unstimulated stimulated unstimulated stimulatedunstimulated stimulated #1 0.14 0.29 0.69 1.25 NT NT #2 0.02 0 0 0.11 NTNT #3 0 0 0.11 1.14 NT NT #4 NT NT 0.05 4.00 0.02 0.48 #5 0.01 0 0.060.36 NT NT #6 0 0.01 0 4.20 NT NT #7 0.02 0.14 0.01 0.42 0.12 0.36 #80.02 0.33 0.33 0.98 NT NT #11 0.3 1.00 0.7 1.30 0.05 0.52 #12 0.04 0.110.10 0.98 NT NT #13 0 0 0 0.88 0.32 0.48 #15 0 0.04 0.05 0.45 NT NT #180 0 0.06 0.62 NT NT #19 0 0.03 0 0.73 NT NT

To assess the functionality of TERT_(572Y)-specific CD8⁺ cells,TERT_(572Y) tetramer-positive cells from in vitro-stimulated PBMCs frompatient #4 (FIG. 4A) were sorted, amplified with PHA and tested fortheir capacity to specifically respond to TERT_(572Y) peptide and tokill TERT-overexpressing tumor cells. More than 90% of amplified cellswere labeled with TERT_(572Y) tetramer (FIG. 4A). PurifiedTERT_(572Y)-specific cells were fully functional, as they produced IFNγand showed CD107a upregulation upon activation with TERT_(572Y) peptide(FIG. 4B). CTL recognized endogenous TERT and specifically killedTERT-transfected but not untransfected N418 fibroblasts (FIG. 4C).Importantly, CTLs killed tumor cells overexpressing TERT (Na8 cells) butnot tumor cells expressing TERT at a low level (ME290 cells) (FIG. 4D).

1.3. Discussion

The aims of the present clinical trial were to evaluate the toxicityprofile and to prove the concept that cryptic peptides derived fromuniversal tumor antigens can induce immunity in cancer patients and can,therefore, be considered for tumor immunotherapy. The inventors used thecryptic peptide TERT₅₇₂, that is presented by HLA-A*0201 and is derivedfrom TERT, a universal tumor antigen overexpressed by 85% of tumors.Immunogenicity had been enhanced by substituting the first amino acid bya tyrosine (Tourdot, Scardino et al. 2000). The results showed thatTERT_(572Y) vaccination of patients with advanced cancer stimulatesspecific CTLs that are fully functional and are able to killTERT-overexpressing tumor cells in vitro. Vaccination was well toleratedand did not appear to induce autoimmunity against TERT-expressing normaltissues. These results offer the first human in vivo confirmation thatoptimized cryptic peptides are good candidates for tumor immunotherapy.

Tumor antigens are non mutated self proteins also expressed by normaltissues, including the thymus, and are involved in tolerance induction.Tolerance, the process by which CTL, mainly those with high-avidity, arepurged from the T cell repertoire, is a major barrier hindering thedevelopment of effective antitumor T cell responses. However, tolerancemainly shapes the T cell repertoire specific for dominant rather thancryptic peptides (Cibotti, Kanellopoulos et al. 1992; Moudgil andSercarz 1994). Using a humanized mouse model, it was recently showedthat vaccination with two cryptic peptides derived from murine TERT(TERT_(572Y) and TERT_(988Y)) recruited high-avidity CTLs capable ofeliciting potent antitumoral immunity (Gross, Graff-Dubois et al. 2004).In the present clinical study, more than 90% of vaccinated patientsdeveloped specific T cells capable of killing TERT-overexpressing tumorcells. In contrast, only 50% of patients treated with the dominantpeptide TERT₅₄₀ emulsified in Monatanide responded to the vaccine(Parkhurst, Riley et al. 2004). However, the natural processing of thedominant TERT540 described initially (Vonderheide, Hahn et al. 1999;Minev, Hipp et al. 2000; Vonderheide, Domchek et al. 2004) was notconfirmed in more recent studies (Ayyoub, Migliaccio et al. 2001;Parkhurst, Riley et al. 2004), which suggests that possibly TERT540 doesnot belong to the immunological self. Given this ambiguity regarding thepresentation of the dominant TERT540 peptide, a direct randomizedcomparison with the cryptic peptide could produce results which would bevery difficult to interpret.

The vaccine response rate in the patients in the present study is higherthan that obtained in the roughly fifty clinical studies of tumorvaccination reported to date (Pullarkat, Lee et al. 2003; Slingluff,Petroni et al. 2003). It is also noteworthy, that almost all previousclinical studies showing high immune response rates involved patientswith minimal disease and excellent performance status (Disis, Gooley etal. 2002; Pullarkat, Lee et al. 2003; Disis, Schiffman et al. 2004;Vonderheide, Domchek et al. 2004). Scheibenbogen et al (Scheibenbogen,Lee et al. 1997) demonstrated that immune reactivity in melanomapatients correlated with disease remission. In contrast, in the presentstudy, all patients had end-stage disease.

No correlation was found between the magnitude of the immune responseand the dose of peptide administered. This is in agreement with recentdata indicating that immune responses to HER2/neu vaccines did notdepend on the vaccine dose (Disis, Schiffman et al. 2004). Nocorrelation was found either between the vaccine dose and the timeinterval required for a detectable response to emerge. All 13 respondingpatients had detectable specific CTLs between the 2^(nd) and the 4^(th)vaccine injection. Rapid induction of immunity may be important in thissetting, especially for patients with rapidly progressive malignancies.

The rationale for using native TERT₅₇₂ peptide for the 3^(rd) to the6^(th) vaccine injections was to select, among T cells recruited by theoptimized TERT_(572Y), those with the highest specificity for TERT₅₇₂presented by tumor cells. Indeed, Clay et al (Clay, Custer et al. 1999)have shown that vaccination of melanoma patients with optimizedgp100_(209M), amplified T cells that were no longer able to recognizeeither the native gp100₂₀₉ peptide or gp100-expressing melanoma cells.The above results show that injection of the native peptide can maintainthe immune response initiated by the optimized peptide. Moreover, thepersistence of the immune response more than one year after the end ofvaccination suggests that native peptide presented on the surface oftumor cells can maintain the specific immune response by itself. Thehallmark of antitumoral immunity in vivo is autoimmunity. Autoimmunityis acceptable when it targets non essential normal cells and tissuessuch as melanocytes, but may hamper vaccine development when it targetsessential cells such as hematopoietic precursors. Although TERT isexpressed by hematopoietic stem cells, gut, thymus, and activated B andT cells (Ramakrishnan, Eppenberger et al. 1998; Liu, Schoonmaker et al.1999), none of the patients showed signs of autoimmunity even 24 monthsafter the end of vaccination. This confirms previous results obtained inHLA-A*0201 transgenic HHD mice vaccinated with TERT_(572Y) peptide,which is also part of murine TERT (Gross, Graff-Dubois et al. 2004).Vaccinated HHD mice developed antitumor immunity without signs ofautoimmunity. Moreover, TERT_(572Y)-specific CTLs killed tumor cells butnot activated B cells. A possible explanation is that TERT expression isinsufficient on normal cells (contrary to tumor cells) to permit thepresentation of low-affinity peptides like TERT₅₇₂.

The observed toxicity in the present study was essentially minimal andwith the exception of transient skin reactions caused by the Montanideadjuvant, all the other mild toxicities could also be attributed to theunderlying disease. Given the limitations of the small number ofpatients enrolled in this trial and the relatively short follow up dueto the advanced disease, it can be concluded that this vaccinationprogram is free of any major acute and short-term toxicity. However,long-term toxicities will have to be evaluated in patients with betterprognosis who are more likely to be cured of their malignant disease.

For ethical reasons, this study involved patients with end-stage cancer,who are not the best candidates for tumor immunotherapy. It is nowgenerally agreed that immunotherapy is best administered to patientswith minimal residual disease, and the goal should be to prevent relapserather than to cure advanced cancer. The inability of vaccines toeradicate actively growing tumors has been clearly shown in animalsmodels (Cheever and Chen 1997). Although clinical antitumor activity bymeans of tumor shrinkage was not observed in this heavily pretreatedgroup of patients, four patients showed long lasting diseasestabilization in this phase I trial. These patients had previouslyprogressive disease and developed TERT-specific CTL which could bedetected in their blood even months after completing the vaccinationprogram. It is interesting that two of these patients (#9 and #13), bothwith renal cell carcinoma, had been successfully treated in the pastwith IL2 or IFNα, confirming the sensitivity of this cancer toimmunotherapy. In contrast, none of 11 patients with renal cancer whowere vaccinated with the dominant TERT₅₄₀ peptide had an objectiveclinical response, even when they developed a peptide-specific immuneresponse (Parkhurst, Riley et al. 2004).

In conclusion, this study demonstrates that vaccination of advancedcancer patients with the optimized cryptic TERT_(572Y) peptide is safeand induces an antitumor immunity in more than 90% of patients. This isthe first clinical confirmation that cryptic peptides are promisingcandidates for cancer immunotherapy.

Example 2 In Vitro Selection and Amplification of CTLs with a HighAvidity for the Native Cryptic Peptide 2.1. Materials and MethodsPeptides

The peptides TERT_(988Y) (YLQVNSLQTV, SEQ ID No: 4), TERT₉₈₈(DLQVNSLQTV, SEQ ID No: 3), MAGE-A_(248V9) (YLEYRQVPV, SEQ ID No: 6) andMAGE-A_(248D9) (YLEYRQVPD, SEQ ID No: 5) have been produced by Epytop(Nimes, France).

Animals and Cells

The HLA-A*0201 transgenic HHD mice and the murine RMAS/HHD tumor cellswere previously described (Pascolo, Bervas et al. 1997).

Generation of CTL in HHD Mice

HHD mice were injected subcutaneously with 100 μg of nonamer peptidesemulsified in incomplete Freund's adjuvant (IFA) in the presence of 150μg of the I-Ab restricted HBVcore128 T-helper epitope. Spleen cells(5×10⁷ cells in 10 ml) from immunized HHD mice were stimulated in vitrowith peptide (10 μM) in RPMI1640+10% FCS for five days. The CTL lineswere established by weekly re-stimulation in vitro with irradiatedspleen cells in the presence of peptide and 50 U/ml IL-2 (Proleukin,Chiron Corp., Emeryville, Calif.).

Cytotoxic Assay

Murine RMAS/HHD cells were used as targets for cytotoxicity as described(Tourdot, Scardino et al. 2000). Briefly, 2.5×10³ ⁵¹Cr-labeled targetswere pulsed with increasing doses (0.00001-10 μM) of peptides at 37° C.for 60 min. Effector cells in 100 μl were then added and incubated at37° C. for 4 hours. After incubation, 100 μl of supernatant werecollected and radioactivity was measured in a gamma counter. Thespecific lysis was determined as:

Lysis=(Experimental Release−Spontaneous Release)/(MaximalRelease−Spontaneous Release).

CTL avidity is defined as the peptide concentration that gives half themaximal lysis. Hence, the lower the measured avidity (in nM), the higherthe avidity of the CTLs.

In Vivo Tumor Protection Assay

HHD mice were vaccinated with 100 μg of peptide emulsified in IFA in thepresence of 150 μg of the 1 Ab restricted HBVcore128 epitope once andthen again two weeks later. One week after the second vaccination theywere challenged subcutaneously with 2×104 EL4/HHD cells. Survival wasrecorded every two days.

2.2. Results

Results Obtained with a Cryptic Epitope from the TERT Antigen

HHD mice were immunized with the optimized cryptic TERT_(988Y) peptide.Eleven days later, spleen cells from vaccinated mice were pooled and invitro serially stimulated with 10 μM of either TERT_(988Y) or the nativecryptic TERT₉₈₈ peptides in the presence of 50 IU/ml of IL2.Stimulations were repeated every week. After the first, third and sixthin vitro stimulation, CTL lines were tested for their avidity for thenative TERT₉₈₈ peptide in a classical ⁵¹Cr release cytotoxicity assay(Table 5).

TABLE 5 Avidity of CTL lines for the native TERT₉₈₈ peptide, establishedfrom TERT_(988Y) primed spleen cells in vitro stimulated with eitherTERT_(988Y) or TERT₉₈₈ peptides Peptide used for No of in vitro in vitroCTL line avidity stimulations stimulation (nM) 1 TERT₉₈₈  170TERT_(988Y) 300 3 TERT₉₈₈  70 TERT_(988Y) 500 6 TERT₉₈₈  3 TERT_(988Y)600Results Obtained with a Cryptic Epitope from the MAGE Antigen

HHD mice were immunized with the optimized MAGE-A_(248V9) peptide,corresponding to the cryptic MAGE-A_(248D9) (both described inWO03083124). Eleven days later, spleen cells from vaccinated mice werepooled and in vitro serially stimulated with 10 μM of eitherMAGE-A_(248V9) or the native cryptic MAGE-A_(248D9) peptides in thepresence of 50 IU/ml of IL2. Stimulations were repeated every week.After the first, third and sixth in vitro stimulation, CTL lines weretested for their avidity for the native MAGE-A_(248D9) peptide in aclassical ⁵¹Cr release cytotoxicity assay (Table 6).

TABLE 6 Avidity of CTL lines established from MAGE-A_(248V9) primedspleen cells in vitro stimulated with either MAGE-A_(248V9) orMAGE-A_(248D9) peptides Peptide used for No of in vitro the in vitrostimulations stimulation CTL avidity (nM) 1 MAGE-A_(248D9) 200MAGE-A_(248V9) 400 3 MAGE-A_(248D9) 40 MAGE-A_(248V9) 450 6MAGE-A_(248D9) 0.8 MAGE-A_(248V9) 320

2.3. Conclusion

Vaccination with optimized cryptic peptides recruits a CTL repertoirethat contains cells with high avidity for the native cryptic peptide.These high avidity CTL can be in vitro selected and amplified bystimulation with the native rather than with the optimized crypticpeptide.

Example 3 In Vivo Selection of CTLs with High Avidity by Boosting withthe Native Peptide 3.1. Materials and Methods

The same materials and methods as in Example 2 were used.

3.2. Results

HHD mice were vaccinated with the optimized cryptic TERT_(572Y) peptidedescribed above. Fifteen days later, they were boosted with either thesame optimized or the native cryptic TERT₅₇₂ peptide. Seven days afterthe boost, their spleen cells were in vitro stimulated with 10 μM of theTERT₅₇₂. CTL generated after one cycle of in vitro stimulation weretested for their avidity for TERT₅₇₂. Table 7 presents results from sixindividual mice.

TABLE 7 Avidity of CTL generated in HHD mice primed with the optimizedcryptic TERT_(572Y) peptide and boosted with either the same optimizedor the native TERT₅₇₂ peptide. Mouse 1st vaccination 2nd vaccination CTLavidity (nM) 1 TERT_(572Y) TERT_(572Y) 350 2 TERT_(572Y) TERT_(572Y) 7003 TERT_(572Y) TERT_(572Y) 650 1 TERT_(572Y) TERT₅₇₂  110 2 TERT_(572Y)TERT₅₇₂  190 3 TERT_(572Y) TERT₅₇₂  70

3.3. Conclusion

In vivo priming with the optimized cryptic peptides recruits arepertoire containing CTL with high avidity for the native peptide.These high avidity CTLs can be in vivo selected and amplified byboosting with the native rather than with the optimized peptide.

Example 4 Tumor Immunity in HHD Mice 4.1. Materials and Methods

The same materials and methods as in Example 2 were used.

An additional peptide, named gp100₁₅₄, was also used: KTWGQYWQV (SEQ IDNO: 8).

4.2. Results

HHD mice were vaccinated with the optimized cryptic TERT_(572Y) andTERT_(988Y) peptides and fifteen days later boosted with either the sameoptimized or the corresponding native cryptic peptide. Ten days afterthe boost, mice were challenged with EL4/HHD tumor cells and monitoredfor tumor growth and survival. Mice primed and boosted with the gp100₁₅₄peptide were used as negative controls (Table 8). 100% of control micedied by day 43 post-challenge. 17% of mice primed and boosted with theoptimized peptides and 50% of mice primed with the optimized and boostedwith the native peptide were definitively protected against tumor.

TABLE 8 Survival of HHD mice primed with the optimized and boosted witheither the same optimized or the corresponding native peptides. Survivalpost- Prime Boost challenge (days) Gp100₁₅₄ Gp100₁₅₄ 41 40 39 39 43 41TERT_(572Y) TERT_(572Y) 31 40 40 40 200+ 45 TERT_(572Y) TERT₅₇₂ 200+200+ 32 35 200+ 40 TERT_(988Y) TERT_(988Y) 200+ 40 40 40 43 43TERT_(988Y) TERT₉₈₈ 39 40 41 200+ 200+ 200+

4.3. Conclusion

Tumor immunity is much more efficient in mice primed with the optimizedand boosted with the native peptide than in mice primed and boosted withthe same optimized peptide.

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1. A vaccination kit, comprising at least one dose of a native peptideand at least one dose of an optimized immunogenic peptide derived fromsaid native peptide, wherein said native and optimized peptides areselected from the group consisting of the following pairs of peptides:TERT572 (SEQ ID No: 1) and TERT572Y1 (SEQ ID No: 2); TERT988 (SEQ ID No:3) and TERT988Y1 (SEQ ID No: 4); MAGE-A248D9 (SEQ ID No: 5) andMAGE-A248V9 (SEQ ID No: 6); MAGE-A248G9 (SEQ ID No: 7) and MAGE-A248V9(SEQ ID No: 6); Gp100 154 (SEQ ID No: 8) and Gp100 154Y1 (SEQ ID No: 9);Gp100 154 (SEQ ID No: 8) and Gp100 154M155 (SEQ ID No: 10); FluM58 (SEQID No: 11) and FluM58Y1 (SEQ ID No: 12); HIVgag76 (SEQ ID No: 13) andHIVgag76Y1 (SEQ ID No: 14); HBVpol575 (SEQ ID No: 15) and HBVpol575Y1(SEQ ID No: 16); HBVpol765 (SEQ ID No: 17) and HBVpol765Y1 (SEQ ID No:18); Mart-127 (SEQ ID No: 19) and Mart-127Y1 (SEQ ID No: 20); Mart-126(SEQ ID No: 21) and Mart-126L27 (SEQ ID No: 22); Gp100 177 (SEQ ID No:23) and Gp100 177Y1 (SEQ ID No: 24); Gp100 178 (SEQ ID No: 25) and Gp100178Y1 (SEQ ID No: 26); Gp100 570 (SEQ ID No: 27) and Gp100 570Y1 (SEQ IDNo: 28); Gp100 209 (SEQ ID No: 29) and Gp100 209Y1 (SEQ ID No: 30);Gp100 209 (SEQ ID No: 29) and Gp100 209M210 (SEQ ID No: 31); Gp100 476(SEQ ID No: 32) and Gp100 476Y1 (SEQ ID No: 33); Gp100 457 (SEQ ID No:34) and Gp100 457Y1 (SEQ ID No: 35); HER-2/neu799 (SEQ ID No: 36) andHER-2/neu799Y1 (SEQ ID No: 37); HER-2/neu369 (SEQ ID No: 38) andHER-2/neu369Y1 (SEQ ID No: 39); HER-2/neu789 (SEQ ID No: 40) andHER-2/neu789Y1 (SEQ ID No: 41); HER-2/neu48 (SEQ ID No: 42) andHER-2/neu48Y1 (SEQ ID No: 43); HER-2/neu773 (SEQ ID No: 44) andHER-2/neu773Y1 (SEQ ID No: 45); HER-2/neu5 (SEQ ID No: 46) andHER-2/neu5Y1 (SEQ ID No: 47); HER-2/neu851 (SEQ ID No: 48) andHER-2/neu851Y1 (SEQ ID No: 49); HER-2/neu661 (SEQ ID No: 50) andHER-2/neu661Y1 (SEQ ID No: 51); HER-2/neu650 (SEQ ID No: 52) andHER-2/neu650Y1 (SEQ ID No: 53); HER-2/neu466 (SEQ ID No: 54) andHER-2/neu466Y1 (SEQ ID No: 55); HER-2/neu402 (SEQ ID No: 56) andHER-2/neu402Y1 (SEQ ID No: 57); HER-2/neu391 (SEQ ID No: 58) andHER-2/neu391Y1 (SEQ ID No: 59); HER-2/neu971 (SEQ ID No: 60) andHER-2/neu971Y1 (SEQ ID No: 61); HBVpol28 (SEQ ID No: 62) and HBVpol28Y1(SEQ ID No: 63); HBVpol594 (SEQ ID No: 64) and HBVpol594Y1 (SEQ ID No:65); HBVpol985 (SEQ ID No: 66) and HBVpol985Y1 (SEQ ID No: 67); EphA2 61(SEQ ID No: 68) and EphA2 61Y1 (SEQ ID No: 69); HER2 911 (SEQ ID No: 70)and HER911Y1V10 (SEQ ID No: 71); HER4 911 (SEQ ID No: 72) andHER911Y1V10 (SEQ ID No: 71); HER1 911 (SEQ ID No: 73) and HER911Y1V10(SEQ ID No: 71); HER2 722 (SEQ ID No: 74) and HER722Y1V9 (SEQ ID No:75); HER3 722 (SEQ ID No: 76) and HER722Y1V9 (SEQ ID No: 75); HER4 722(SEQ ID No: 77) and HER722Y1V9 (SEQ ID No: 75); HER1 722 (SEQ ID No: 78)and HER722Y1V9 (SEQ ID No: 75); HER2 845 (SEQ ID No: 79) and HER845Y1(SEQ ID No: 80); HER3 845 (SEQ ID No: 81) and HER845Y1 (SEQ ID No: 80);HER2 904 (SEQ ID No: 82) and HER904Y1 (SEQ ID No: 83); HER4 904 (SEQ IDNo: 84) and HER904Y1 (SEQ ID No: 83); HER2 933 (SEQ ID No: 85) andHER933Y1 (SEQ ID No: 86); HER1 933 (SEQ ID No: 87) and HER933Y1 (SEQ IDNo: 86); HER2 945 (SEQ ID No: 88) and HER945Y1 (SEQ ID No: 90); HER3 945(SEQ ID No: 89) and HER945Y1 (SEQ ID No: 90); HER4 945 (SEQ ID No: 91)and HER945Y1 (SEQ ID No: 90); and HER1 945 (SEQ ID No: 92) and HER945Y1(SEQ ID No: 90), and wherein said vaccination kit further comprises anotice of use indicating that the kit is intended for performing amethod of vaccinating a patient against a tumoral or viral antigencomprising a first step of vaccination with an optimized peptide cognateto a native peptide of said antigen, followed by a second step ofvaccination with said native peptide, whereby the step of vaccinationwith the native peptide amplifies CTLs with high avidity for said nativepeptide.
 2. The vaccination kit of claim 1, which comprises 2 or 3 dosesof optimized peptide, and 3, 4, 5, 6 or up to 50 doses of nativepeptide.
 3. The vaccination kit of claim 1, wherein each dose comprises1 to 5 mg of peptide.
 4. The vaccination kit of claim 1, wherein saidvaccination doses are formulated for subcutaneous injection.
 5. Thevaccination kit of claim 1, wherein said vaccination doses areformulated for a human patient.
 6. The vaccination kit of claim 1,wherein said native peptide is from a tumor antigen.
 7. The vaccinationkit of claim 1, wherein said native peptide is from a viral antigen.